Loss of sensitivity to TGF-beta-dependent signaling is an important contributing factor in pancreatic tumor development. Deletions or mutations in Smad4, a key downstream determinant in TGF-beta signaling, have been identified in about 50% of pancreatic adenocarcinomas, and protein instability of Smad4 mutants is thought to play an important role in the loss in cellular responsiveness to TGF-beta in pancreatic tumorigenesis. Studies of the mechanism underlying the degradation of Smad4 in pancreatic carcinoma should be instructive for further understanding of the pathogenesis of this disease. In our preliminary studies, we have identified ubiquitin E3 ligase SCFbeta-TrCP1 as a critical determinant for the protein degradation of Smad4. We found that: 1) F-box protein beta-TrCP1 in this E3 ligase interacts with Smad4 in yeast and in mammalian cells. MH1 plus Linker region of Smad4 is the domain that mediates the interaction. 2) SCF beta-TrCP1 efficiently induces the ubiquitination and degradation of Smad4.3) SCFbeta-TrCP1 inhibits TGF-beta-induced gene transactivation, cell cycle arrest and apoptosis. 4) Pancreatic cancer cell lines harboring Smad4 point mutation exhibited much higher Smad4 ubiquitination and protein instability mediated by SCFbeta-TrCP1. 5) Using siRNA-induced beta-TrCP1 gene silencing, the expression level of Smad4 protein was elevated, and TGF-beta-induced gene transcription in pancreatic cancer cell lines was rescued. Therefore, we hypothesize that beta-TrCP1 is a key molecule that controls the protein stability of tumor suppressor Smad4, and increased Smad4 protein level by beta-TrCP1 gene silencing rescues the loss of TGF-beta responses in pancreatic cancer cells. We will pursue the following specific aims: 1) Determine the mechanisms by which beta-TrCP1 recognizes Smad4 and mediates its degradation. 2) Determine the effects of beta-TrCP1 gene silencing on Smad4 protein stability and TGF-beta-mediated cellular responsiveness in pancreatic cancer cells. We believe that characterization of Smad4 degradation by SCFbeta-TrCP1 would provide important new insights into the mechanism responsible for the inactivation of tumor suppressor Smad4 in pancreatic cancer, and also enable development of a potential therapeutic approach for pancreatic carcinoma.